Cellular functions are controlled by sophisticated signal transduction pathways triggered by receptors responding to myriad environmental stimuli. With the rise of synthetic biology, we can now engineer artificial receptors enabling real-time interrogation and manipulation of cellular signaling, and providing new clues about the design principles of natural sensing systems. In this review, we describe the main classes of synthetic receptors engineered to date, their applications, and highlight recent developments that might improve synthetic receptor design in the future. © 2020 Elsevier Inc. All rights reserved.Nuclease-mediated DNA cleavage and subsequent repair lie at the heart of genome editing, and the RNA-guided endonuclease Cas9 has emerged as the most widely-used tool for facilitating this process. Extensive biochemical and biophysical efforts have revealed much regarding the structure, mechanism, and cellular properties of Cas9. This has enabled engineering of Cas9 variants with enhanced activity, specificity, and other features. However, we lack a detailed understanding of the kinetics of Cas9-mediated DNA cleavage and repair in vivo. To study in vivo Cas9 cleavage kinetics and activity dose-dependence, we have engineered a chemically-inducible, single-component Cas9, ciCas9. ciCas9 allows for temporal and rheostatic control of Cas9 activity using a small molecule activator, A115. We have also developed a droplet-digital PCR-based assay (DSB-ddPCR) to directly quantify Cas9-mediated double-stranded breaks (DSBs). The methods in this chapter describe the application of ciCas9 and DSB-ddPCR to study the kinetics and dose-dependence of Cas9 editing in vivo. © 2020 Elsevier Inc. All rights reserved.RAS GTPases are involved in a number of dynamic signaling processes and have been a major focus of research due to the prevalence of activating RAS mutations in cancer. However, despite decades of research, some fundamental aspects of RAS biology are still not well understood. Difficulty in fully defining RAS-driven signaling stems from the overall complexity of downstream pathways and a lack of tools for specifically perturbing RAS function. To better characterize RAS-driven signaling, we recently developed a chemical genetic system for activating endogenous RAS with a small molecule. In this chapter, we describe the use of chemically inducible activator of RAS (CIAR), a single-protein, chemical genetic system that allows the rapid and dose-dependent activation of endogenous RAS. Methods in this chapter also describe the validation of RAS activation with CIAR through the analysis of downstream signaling. © 2020 Elsevier Inc. All rights reserved.Biotinylated molecules are extensively employed in bioanalytics and biotechnology. The currently available assays for quantification of biotin groups suffer from low sensitivity, low accuracy, or provide highly variable responses for different biotin derivatives. We developed a competitive binding assay in which avidin was pre-blocked to different extents by the biotinylated analyte and a constant amount of biotin-4-fluorescein (B4F) was added, resulting in strong quenching of the B4F. The assay was robust and the shape of the titration curve immediately revealed whether the data were reliable or perturbed by steric hindrance in case of large biotin derivatives. These advantages justified well the 10× higher sample consumption (~0.6nmol) compared to single point assays. The assay was applied to a representative set of small biotin derivatives and validated by cross-control with the well-established 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) binding assay. In comparison to the 2,6-ANS binding assay, the lower precision (±10%) was compensated by the 100-fold higher sensitivity and the deviations from the ANS assay were ?5%. In comparison to the more sensitive biotin group assays, the new assay has the advantage of minimal bias for different biotin derivatives. In case of biotinylated DNA with 30 nucleotides, steric hindrance was found to reduce the accuracy of biotin group determination; this problem was overcome by partial digestion to n?5 nucleotide residues with a 3'-exonuclease. The newly proposed biotin group assay offers a useful compromise in terms of sensitivity, precision, trueness, and robustness. © 2020 Elsevier Inc. All rights reserved.PURPOSE The purpose of this study was to compare primary outcomes following insertion of balloon and nonballoon gastrostomy tubes (G-tubes). METHODS A retrospective chart review over a 5-year period comparing the need for emergency, radiologic, or operative interventions between balloon and nonballoon G-tube devices was performed. RESULTS 145 patient charts were reviewed (46.8% female, 53.1% male). The indication for G-tube insertion was failure to thrive in 83.4%. https://www.selleckchem.com/products/azd5153-6-hydroxy-2-naphthoic-acid.html Average age at insertion was 4.3?years (0-17.9?years). 37.2% had a balloon type G-tube, and 62.8% had a nonballoon type. Patients with a nonballoon device had 1.14 (0-15) ER visits related to the G-tube vs. 0.48 (0-6) visits with a balloon device. Of the ER visits for patients with a nonballoon device, 26.9% were replaced in ER, 38.5% in radiology, and 34.6% required an operation for replacement. For patients with a balloon device, 47.8% were replaced in the ER, 52.2% were replaced in radiology (GJ), and none required operative replacement. The majority of patients who initially had a nonballoon G-tube placed required a second operation for device change (95.7%). Patients with nonballoon devices required significantly more operations (average 2.55, range 0-16) vs patients with balloon devices (average 0.40, range 0-3) (p? less then ?.05). CONCLUSIONS Balloon-type G-tubes require less ER visits and operative interventions compared to nonballoon G-tubes. LEVEL OF EVIDENCE C. Various definitions and biomarkers have been developed in an unsuccessful attempt to obtain a "gold standard" for periprosthetic joint infection (PJI) diagnosis. The development of the 2011 Musculoskeletal Infection Society criteria facilitated further research and advances by allowing the use of a consistent PJI definition across studies. The newly proposed 2018 criteria do not rely at all on expert opinions/consensus. In this review, we describe the most relevant definitions developed throughout recent time, their rationale, characteristics, and supportive evidence for their clinical implementation. In the opinion of the authors, the orthopedic community should consider a probability and likelihood paradigm to create a PJI diagnostic definition. Probably not a single definition might be suited for all situations; the inclusion of serological findings could be the next step moving forward.